PROJECT SUMMARY Epidemic keratoconjunctivitis (EKC), an important human eye infection, is caused by human adenovirus species D (HAdV-D) types 8, 37, 53, 54, 56, and 64. The clinical manifestations of this infection include severe membranous conjunctivitis and epithelial keratitis, followed by multifocal subepithelial (stromal) corneal infiltrates that cause photophobia and reduced vision and may persist or recur for months to years after the acute infection. We have shown previously that HAdV-D infection of human keratocytes induces intracellular signaling and robust expression of neutrophil chemokines, and that physical components of the adenovirus (viral capsid and DNA) initiate distinct cytokine responses in the corneal stroma. However, characterization of responses to infection of other ocular surface cell types such as corneal and conjunctival epithelial cells, is lacking,. The specific aims of this proposal are to test the hypotheses that 1) HAdV-D37 infection of ocular surface cells induces cytokine signatures distinguishable by cell type, 2) HAdV-D37 infection of ocular surface cells activates unique molecular pattern recognition receptors distinguishable by cell type, and 3) cell type-specific pattern recognition receptor activation by HAdV-D37 and intracellular trafficking of HAdV-D37 are interdependent. We will apply intracellular cytokine staining with flow cytometry along with cytokine protein array analysis of the various cell types on the human ocular surface to identify cell type-specific patterns of cytokine expression upon HAdV-D37 infection. Because CXCL8 has been shown to increase subsequent adenoviral entry by increasing the expression and apical localization of host cell receptors, we will compare infectivity of corneal cells in the presence of cytokines expressed during infection of conjunctival and immune cells from the ocular surface. We will apply Western blot, immuno-confocal microscopy, ELISA, cytokine arrays, and siRNA knockdown, to determine the molecular pattern receptors activated in human ocular surface and bone marrow derived cells by HAdV-D37 infection. Finally, with methods that allow differentiation of intact virus versus capsid-free viral DNA and endosomal versus cytoplasmic virus, with confocal microscopy, we will characterize viral trafficking and its kinetics across ocular surface cell types, identify loci of interaction between viral DNA and specific pattern recognition receptors, and evaluate the effect of pattern recognition receptor knockdown on viral trafficking. The study of cytokine expression, pattern recognition receptor activation and signaling, and intracellular trafficking across a broad spectrum of ocular surface cell types, will take us closer to our goal of effective information-based therapies against HAdV eye infections. EKC is a common affliction, and the proposed study addresses a major public health concern.